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Cyclotides are a new emerging family of large plant-derived backbone-cyclized polypeptides (≈30 amino acids long) that share a disulfide-stabilized core (3 disulfide bonds) characterized by an unusual knotted structure. Their unique circular backbone topology and knotted arrangement of three disulfide bonds makes them exceptionally stable to thermal, chemical and enzymatic degradation compared with other peptides of similar size. Currently more than 100 sequences of different cyclotides have been characterized and the number is expected to increase dramatically in the coming years. Considering their stability and biological activities like anti-HIV, uterotonic and insecticidal; and also their abilities to cross the cell membrane, cyclotides can be exploited to develop new stable peptide-base drugs. We have recently demonstrated the intriguing possibility of producing libraries of cyclotides inside living bacterial cells. This opens the possibility to generate large genetically-encoded libraries of cyclotides that can then be screened inside the cell for selecting particular biological activities in a high-throughput fashion. The present mini-review reports the efforts carried out toward the selection of cyclotide-based compounds with specific biological activities for drug design.
Peptides have proven to be valuable and effective drugs when targeting protein-protein interactions.1,2 The concept of using peptides to modulate intracellular processes has been investigated for decades, as peptides play a central role in every cell in the body. Polypeptides that mimic protein fragments typically are able to provide effective competitive binding antagonists for protein-protein interactions. Targeting these interactions which, usually involve large binding surfaces has proven challenging for small molecules. The utility of peptide therapeutics, however, has typically been limited by their generally poor stability and limited bioavailability. For example, linear peptides are typically inherently unstable within the body and are rapidly broken down into inactive fragments by proteolytic enzymes, which are then filtered from the blood stream by the kidneys within minutes. In response to this challenge a number of novel polypeptide scaffolds are starting to emerge to replace classical peptide and protein-based therapeutics.2–9
This paper reviews the latest developments on the use of cyclotides as a molecular scaffold for delivering a novel type of peptide-based therapeutics with the specificity of proteins but the physical-chemical properties that are usually associated with small molecule-based drugs.
Special attention has been recently given to the use of highly constrained peptides, also known as micro- or mini-proteins, as extremely stable and versatile scaffolds for the production of high affinity ligands for specific protein capture and/or development of therapeutics.5,8 Cyclotides are fascinating micro-proteins (≈30 amino acids long) present in plants from the Violaceae, Rubiaceae and also Cucurbitaceae; and featuring various biological actions such as protease inhibitory, insecticidal, cytotoxic, anti-HIV or hormone-like activity. 10,11 More recently, it has been demonstrated that cyclotides also show potent anthelmintic activity.12,13 Their insecticidal and anthelmintic properties suggest that they may function as defense molecules in plants.
Cyclotides share a unique head-to-tail circular knotted topology of three disulfide bridges, with one disulfide penetrating through a macrocycle formed by the two other disulfides and inter-connecting peptide backbones, forming what is called a cystine knot topology (Figure 1). These micro-proteins can be considered as natural combinatorial peptide libraries structurally constrained by the cystine-knot scaffold and head-to-tail cyclization but in which hypermutation of essentially all residues is permitted with the exception of the strictly conserved cysteines that comprise the knot.14–16 The main features of cyclotides are therefore a remarkable stability due to the cystine knot, a small size making them readily accessible to chemical synthesis, and an excellent tolerance to sequence variations. For example, the first cyclotide to be discovered, kalata B1 (kB1), is an orally effective uterotonic17. Intriguingly, the MCoTI-II cyclotide has been also shown to cross the cell membrane through macropinocytosis.18
There have been a number of reports showing the plasticity of the cyclotide framework and its tolerance to substitution. The first proof of concept for grafting of new sequences onto kB1 was established by replacing some hydrophobic residues in loop 5 with charged and polar residues. These residues form part of a surface-exposed hydrophobic patch that plays a significant role in the folding and biological activity of kB1. The modified cyclotide analogs retained the native fold and lacked the undesirable hemolytic activity of the parent peptide despite the importance of these residues.14
Novel VEGF-A antagonists have been also developed by grafting a peptide sequence able to antagonize VEGF-A onto kB1.19 One of the grafted analogs showed biological activity at low micromolar concentration in an in vitro VEGF-A antagonism assay, and the in vitro stability of the target epitope was markedly increased using this approach.
The use of the cyclotide scaffold in drug design has been also recently shown by engineering non-native activities into the cyclotide MCoTI-II (Figure 1).20 Replacing the P1 residue in the active loop of the cyclotide produced several MCoTI-II analogs with different specificities towards alternative protease targets. Interestingly, several analogs showed selective low-μM inhibition of foot-and-mouth-disease virus (FMDV) 3C protease. These are the first reported peptide-based inhibitors of this protease and although the potency was relatively low (low μM), this study demonstrates the potential of using MCoTI-based cyclotides for designing novel protease inhibitors.20 Hence, the MCoTI-cyclotide appears to be a versatile scaffold for the display of biological activities as it has also been shown to be able to cross cellular membranes.18 This opens the possibility of using it for delivering biological active peptide sequences to intracellular targets. Several grafting studies using the MCoTI-cyclotide scaffold to target several intracellular targets involved in programmed cell death and in tumor cell proliferation and suppression are currently underway in our laboratory.
MCoTI cyclotides share a high sequence homology with related cystine-knot trypsin inhibitors found in squash such as EETI-II (Ecballium elaterium trypsin inhibitor II), and in fact can be considered cyclized homologs of the these protease inhibitors. Squash cystine-knot trypsin inhibitors have also successfully been used to graft biological activities. Thus, the RGD sequence, originally discovered in disintegrins, has been grafted into loop 1 of EETI-II yielding an EETI-II analog with platelet inhibitory activity.21 Interestingly, the engineered proteins were much more potent in inhibiting platelet aggregation than the grafted peptides, highlighting the importance of grafting a linear epitope into a stable peptide-scaffold. These highly stable peptides could have clinical use for the treatment of patients with acute coronary syndrome, for example.
In addition to displaying biological activities, EETI-II peptides have also been shown to permeate through rat small intestinal mucose more effectively compared to other peptide drugs such as insulin and bacitracin.22 These analogs also showed comparable stability to cyclotides in plasma, thus supporting evidence for the utility of highly constrained cystine-knot peptides in drug design.
All these data highlight the extraordinary pharmacokinetic properties of cyclotides and cystine-knot peptides in general thus confirming the potential of these polypeptide scaffolds in peptide-based drug discovery.
Cyclotides are ribosomally produced in plants from precursors that comprise between one and three cyclotide domains; however, the mechanism of excision of the cyclotide domains and ligation of the free N- and C-termini to produce the circular peptides has not been completely elucidated yet.23,24
Our group has recently developed and successfully used a bio-mimetic approach for the biosynthesis of folded cyclotides inside bacterial cells by making use of modified protein splicing units in combination with an in-cell intramolecular native chemical ligation reaction (NCL) (Figure 2).16,25,26 Intramolecular NCL requires the presence of an N-terminal Cys residue and a C-terminal α-thioester group in the same linear precursor molecule.27–29 For this purpose, the linear cyclotide precursors were fused in frame at their C- and N-terminus to a modified intein and a Met residue, respectively. This allows the generation of the required C-terminal α-thioester and N-terminal Cys residue after in vivo processing by endogenous Met amino peptidase (MAP). Our group has also recently used this bio-mimetic approach for the biosynthesis of another backbone-cyclized peptide, the Bowman-Birk inhibitor SFTI-1 (sunflower trypsin inhibitor 1);30 and biosynthesis of other cyclic peptides such backbone-cyclized α-defensins and naturally occurring θ-defensins is currently underway in our laboratory.
Recombinant biosynthesis of cyclic polypeptides offers many advantages over purely synthetic methods.29 Using the tools of molecular biology, large combinatorial libraries of cyclic peptides may be generated and screened in vivo. A typical chemical synthesis may generate 104 different molecules. It is not uncommon for a recombinant library to contain as many as 109 members. The molecular diversity generated by this approach is analogous to phage-display technology. The approach, however, differs from phage-display in that the backbone-cyclized polypeptides are not fused to or displayed by any viral particle or protein, but remain on the inside of the living cell where they can be further screened for biological activity in an analogous way as the yeast two hybrid technology works.31 The complex cellular cytoplasm provides the appropriate environment to address the physiological relevance of potential leads.
The ability to create cyclic polypeptides in vivo opens up the possibility of generating large libraries of cyclic polypeptides. Using the tools of molecular biology, genetically encoded libraries of cyclic polypeptides containing billions of members can be readily generated. This tremendous molecular diversity forms the basis for selection strategies that model natural evolutionary processes. Also, since the cyclic polypeptides are generated inside living cells, these libraries can be directly screened for their ability to attenuate or inhibit cellular processes.
We have reported recently the biosynthesis of a genetically encoded library of MCoTI-I based cyclotides in Escherichia coli cells.16 The cyclization/folding of the library was performed either in vitro, by incubation with a redox buffer containing glutathione, or by in vivo self-processing of the corresponding precursor proteins. Out of 27 mutations studied only two mutations, G25P and I20G, negatively affected the folding of cyclotides (Figures 1 and and3).3). These data provide significant insights into the structural constraints of the MCoTI cyclotide framework and the functional elements for trypsin binding. To our knowledge, this is the first time that the biosynthesis of a genetically-encoded library of MCoTI-based cyclotides containing a complete suite of amino acid mutants is reported. Craik and co-workers have also recently reported the chemical synthesis of a complete suite of Ala mutants for kB1.15 These mutants were fully characterized structurally and functionally. Their results indicated that only two of the mutations explored (kB1 W20A and P21A, both located in loop 5, see Figure 1) prevented folding.15 The mutagenesis results obtained in our work show similar results highlighting the extreme robustness of the cyclotide scaffold to mutations. These studies show that cyclotides may provide an ideal scaffold for the biosynthesis of large combinatorial libraries inside living bacterial cells, which can then be screened in-cell for biological activity using high-throughput flow cytometry techniques.32–34
Our group has recently developed a protease cell-based screening reporter using a couple of optimized genetically encoded fluorescent proteins for FRET-based screening.34 We are currently using this approach for screening genetically-encoded libraries based on the MCoTI-I cyclotide scaffold against several proteases, including Anthrax lethal factor.
In summary, cyclotides have several characteristics that make them ideal drug development tools. First, they are remarkably stable due to the cystine knot. Second, they are small, making them readily accessible to chemical synthesis and therefore chemical lead optimization.20,35–37 Third, they can be encoded within standard cloning vectors, and expressed in bacteria or animal cells, and are amenable to substantial sequence variation.16,25,26 Finally, some cyclotides have been shown to be orally bioavailable17 and able to cross the cell membrane through macropinocytosis.18 These characteristics make them ideal substrates for molecular evolution strategies to enable generation and selection of compounds with optimal binding and inhibitory characteristics. Cyclotides thus appear as very promising leads or frameworks for the development of novel peptide-based therapeutics and diagnostics.4,5,8,14
This work was supported by funding from the School of Pharmacy at the University of Southern California and by National Institute of Health award GM090323-01 to J.A.C.